Planococcus notacanthi sp. nov., isolated from the skin of a deep-sea snub-nosed spiny eel

Abstract A novel bacterial strain, APC 4016T, was previously isolated from the skin of a snub-nosed spiny eel, Notacanthus chemnitzii, from a depth of 1000 m in the northern Atlantic Ocean. Cells were aerobic, cocci, motile, Gram-positive to Gram-variable staining, and gave rise to orange-pigmented colonies. Growth occurred at 4–40 °C (optimum, 25–28 °C), pH 5.5–12 (optimum, pH 7–7.5), and 0–12 % (w/v) NaCl (optimum, 1 %). 16S rRNA gene phylogenetic analysis confirmed that strain APC 4016T belonged to the genus Planococcus and was most closely related to Planococcus okeanokoites IFO 12536T (98.98 % 16S similarity). However, digital DNA–DNA hybridization and average nucleotide identity values between these two strains were low, at 20.1 and 83.8 %, respectively. Major (>10 %) cellular fatty acids of strain APC 4016T were iso-C14 : 0, anteiso-C15 : 0 and C16 : 1-ω-Alc. The predominant respiratory quinones were menaquinones 5, 6, 7 and 8. The major cellular polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, and three unknown lipids were also present. The draft genome sequence is 3.6 Mb with a G+C content of 45.25 mol%. This strain was previously shown to have antimicrobial activity and to encode bacteriocin and secondary metabolite biosynthetic gene clusters. Based on the phylogenetic analysis and its distinct phenotypic characteristics, strain APC 4016T is deemed to represent a novel species of the genus Planococcus, and for which the name Planococcus notacanthi sp. nov. is proposed. The type strain of this species is APC 4016T (=DSM 115753T=NCIMB 15463T).


BACKGROUND
The genus Planococcus was first proposed by Migula [1] in order to distinguish motile marine cocci from the genus Micrococcus.It has proven to be a difficult taxonomic group to describe; the genus has undergone several emendations [2,3] and members have been frequently reclassified, mainly between Planococcus and Planomicrobium [4][5][6].Previously, members of the genera Planococcus and Planomicrobium were primarily distinguished based on signature nucleotides of the 16S rRNA gene sequence, particularly those at positions 183 and 190 [7].However, an extensive demarcation of Planococcaceae and related families was recently proposed by Gupta and Patel [8].They carried out extensive phylogenetic and comparative analysis of core protein sequences from 124 complete genomes from Caryophanaceae, Planococcaceae and selected Bacillaceae species.From their analysis, they proposed the consolidation of the families Planococcaceae and Caryophanaceae (under the name Caryophanaceae).They established that sequences from each species could be reliably grouped into distinctive clades, which were distinguished based on their phylogenetic grouping and on the presence of unique molecular markers ('conserved signature indels').These conserved signature indels were specific to either Caryophanaceae/Planococcaceae members or to clades within this family.They also proposed the creation of a novel genus, Metaplanococcus, the reclassification of several species, and that the genera Planococcus and Planomicrobium be united under the name 'Planococcus'.The emended genus Planococcus now includes most species from the genus Planomicrobium.To date, there are 26 valid type strains within the genus Planococcus (Planococcus/Planomicrobium).Species have been isolated from a wide variety of sources, primarily marine or halophilic environments, such as marine sediment [3,5], algal mats [9,10] and seafood [4,11].Others have been found in extreme conditions including sub-zero temperatures [12][13][14], deep-sea sediment [15] and in polluted soils [16,17].Members of the genus Planococcus are of great biotechnological interest because of their robustness and diverse biological activities, such as production of cold-adapted and halotolerant enzymes [18][19][20], radiation tolerance [21], plant growth promotion [22] and antioxidant and cytotoxic activities [23,24].They have also been extensively studied for their potential applications in bioremediation and polysaccharide degradation [25][26][27][28].
Strain APC 4016 T was previously isolated during a study of antimicrobial-producing isolates from the microbiome of deep-sea fish, and subsequently shown to encode secondary metabolites and bacteriocins [29].In this study, we report the taxonomic characterization of a Planococcus bacterial strain, APC 4016 T , isolated from the skin of a deep-sea snub-nosed spiny eel (Notacanthus chemnitzii).

Isolate information
Strain APC 4016 T was previously isolated from the skin of a snub-nosed spiny eel (Notacanthus chemnitzii) [29].In brief, the fish was collected by research vessels from a depth of approximately 1000 m in international waters near the Grand Banks of Newfoundland in the north-western Atlantic Ocean (43.282N 49.121 W) [30].Swabs were taken of the skin and plated onto BD Difco marine agar 2216 (MA) and incubated aerobically for 3 weeks at 4 °C.An orange-coloured colony was selected and purified through sub-culturing on MA.The strain, designated APC 4016, was deposited into the APC Culture Collection, cells of which were preserved in 35 % (v/v) glycerol suspensions at −80 °C [29].This strain has also been deposited into the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (=DSM 115753 T ) and the National Collection of Industrial, Food and Marine Bacteria (= NCIMB 15463 T ).

Genome sequencing, assembly and annotation
Strain APC 4016 T was cultured in BD Difco marine broth 2216 (MB) for 72 h at 20 °C.Bacterial genomic DNA (gDNA) was extracted and sequenced, as previously described [29].Briefly, gDNA was extracted using the GeneJET Genomic DNA Purification Kit (Thermo Scientific) and sequenced by MicrobesNG (https://microbesng.com/; University of Birmingham, UK).The draft genome was assembled using SPAdes (de novo assembly method), and assembly quality was assessed using quast.The assembled contigs were submitted to GenBank and annotated upon submission using NCBI's Prokaryotic Annotation Pipeline.For this study, the draft assembly was annotated using prokka [31].Additionally, functional subsystems were predicted using RASTtk (version 2.0) and seed [32,33].AntiSMASH and bagel4 were used to screen for the presence of secondary metabolite and bacteriocin biosynthetic gene clusters (BGCs), respectively, as previously reported [29].

Genomic phylogeny
For the following analyses, all available Planococcus/Planomicrobium valid type strain reference genomes (n=22) were downloaded from the GenBank (www.ncbi.nlm.nih.gov/data-hub/genome/) and JGI (https://img.jgi.doe.gov/)databases.Assemblies were annotated using prokka to obtain the GFF3 files for subsequent analysis.Pairwise average nucleotide identity (ANI) values were calculated between APC 4016 T and the Planococcus/Planomicrobium type strain reference assemblies with Pyani (version 0.2.12; [42]) using the ANI MUMer (ANIm) method [43].roary [44] was used to generate multiple sequence alignments of the core genes of APC 4016 T and the type strain reference genomes.The annotated genome of Sporosarcina aquimarina S/N-308-OC-B4 was included as an outlier (roary parameters: 95 % 'identity for blast' , 95 % 'percentage of isolates a gene must be in to be core' , 100 000 cluster limit).RaxML [45] was used to generate a maximum-likelihood phylogenetic tree from the core gene alignment, which was then visualized in mega X.Additionally, the DSMZ Type (Strain) Genome Server (TYGS) annotation platform [46] was used to calculate digital DNA-DNA hybridization (dDDH) values between APC 4016 T and related type strains from the database.

Biochemical and phenotypic characterisation
Colony morphology was assessed by incubating aerobically at 25 °C on MA and on tryptic soy agar (TSA; Oxoid/Ther-moFisher Scientific) and observed after 3 and 5 days.Growth at different temperatures was tested at 4, 9, 21, 25, 30, 37, 40 and 44 °C on MA and in MB.For the latter, growth was assessed by measuring optical density at OD 600nm .The following tests were performed on strain APC 4016 T and, unless stated otherwise, were carried out aerobically at 30 °C.Anaerobic growth was assessed on MA incubated in an AnaeroGen anaerobic system (Oxoid) for up to 14 days.The NaCl range for growth was tested in MB with the NaCl concentration adjusted to within the range 0-22 % w/v (in increments of 1 % from 0-10 % NaCl, and in increments of 2 % from 12-22 % NaCl).The pH range for growth was tested in MB from pH 4 to 12 (in increments of 0.5 from pH 4 to 9 and increments of 1 from pH 9 to 11).The pH was adjusted by the addition of 1 M HCl and/or 1 M NaOH.Growth was assessed by measuring optical density at OD 600nm daily for 4 days.Cells were examined using phase contrast microscopy and scanning electron microscopy (SEM).SEM imaging was carried out by UCD Conway Imaging Core (UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland).Gram staining was carried out according to standard methods.Production of oxidase was tested using oxidase strips (Millipore) according to the manufacturer's instructions.Production of catalase was tested by transferring a mass of colonies onto a glass slide and exposing them to 1-2 drops of 3 % hydrogen peroxide and observing for effervescence.Observation for motility was carried out using the hanging drop method according to the method outlined by Bernardet et al. [47] except that a cavity microscope slide was used.The presence of flexirubin-type pigments was tested using the KOH method as described by Bernardet et al. [47].Production of hydrogen sulphide was determined using Watman lead acetate strips (Sigma-Aldrich) suspended above an inoculum of the strain in MB in a test tube and was monitored for up to 7 days.
Artificial seawater (ASW) salts were prepared for the following assays according to Kurilenko et al. [48] as follows: 30 g l −1 NaCl, 5.94 g l −1 MgSO 4 •7H 2 O, 4.53 g l −1 MgCl 2 •6H 2 O, 0.64 g l −1 KCl and 1.3 g l −1 CaCl 2 •2H 2 O. Assimilation of Tween 80 and starch was tested by plating on agar medium (15 g l −1 agar) containing ASW salts, 5 g l −1 peptone, 1 g l −1 yeast extract, 0.1 g l −1 K 2 HPO 4 , and 1 % v/v Tween 80 or 0.2 % w/v starch.Hydrolysis of DNA was determined using DNase Test Agar (Thermo Scientific) supplemented with ASW salts.Hydrolysis of casein was tested on agar medium containing ASW salts supplemented with 10 % w/v skimmed milk powder.Hydrolysis of cellulose was determined by culturing APC 4016 T on agar medium containing ASW salts, 0.2 % w/v cellulose and 0.5 % w/v peptone; following incubation the plate was then stained with 1 % Congo red, washed with 1 M NaCl and observed for zones of clearance around colonies.
Analyses of cellular fatty acids, polar lipids and respiratory quinones was carried out by DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany).For fatty acid and polar lipid analyses, strain APC 4016 T was prepared by culturing on MA at 25 °C.Cellular fatty acids were determined using the Sherlock MIS (midi) system (version 6.1; TSBA40 method, TSBA6 calculation).

Genome features and characteristics
The draft genome assembly of strain APC 4016 T was 3 640 984 bp in length with a G+C content of 45.25 mol%.An overview of the general characteristics of the draft genome is given in Table 1.Using rast, 1603 subsystem features were identified across 24 subsystem categories.An overview of the composition of rast subsystem features is shown in Fig. 1.The largest subsystem categories were dedicated to 'amino acids and derivatives' , at 297 features/genes (18.5 % of the total), followed by 'carbohydrates' , at 254 features (15.8 %) and 'protein metabolism' at 187 features (11.7 %).Three secondary metabolite BGCs were identified using antiSMASH, encoding two terpenes, one of which shared 28 % similarity to the BGC of carotenoid, and a type-3 polyketide synthase (T3PKS) which shared 8 % similarity to beta-lactam.A BGC encoding two lanthipeptides (class II/LanM-associated) was detected using bagel4, the biosynthetic genes of which shared 50 and 55.6 % match with cerecidin (Bacillus cereus), respectively (data from Uniacke-Lowe et al. [29]).An overview of the antiSMASH and bagel screening results is given in Table S1.

16S rRNA gene and genome phylogenetic analysis
The 16S rRNA gene sequence of strain APC 4016 T is 1548 bp in length.Analysis of the 16S rRNA gene sequence using the EzBioCloud search tool indicated that APC 4016 T was most closely related to members of the genus Planococcus (Planococcus/Planomicrobium) within the family Caryophanaceae.The 16S rRNA gene sequence of APC 4016 T showed the highest similarity to Planomicrobium okeanokoites IFO 12536 T (98.98 % similarity) followed by Planococcus plakortidis DSM 23997 T (98.64 %), Planococcus salinarum DSM 23820 T (=ISL-16 T , 98.64 %), Planococcus donghaensis DSM 22276 T (98.57%) and Planococcus halotolerans SCU63 T (98.52 %).Completeness values were all 100 %.These strains were chosen as reference strains for the comparative phenotypic and chemotaxonomic analyses.The maximum-likelihood phylogenetic tree created from the 16S rRNA gene sequences (Fig. 2) shows the evolutionary relatedness between APC 4016 T and the EzBioCloud hits.There was slight variability in topology between the maximum-likelihood, neighbour-joining (Fig. S1, available in the online version of this article) and maximum-parsimony (Fig. S2) trees, likely due to low bootstrap confidence.However, in all three trees, APC 4016 T either formed its own clade or clustered with P. okeanokoites IFO 12536 T .A summary of the overall genome relatedness between APC 4016 T and the reference strains is given in Table 2.The pairwise ANI values between APC 4016 T and the reference Planococcus/Planomicrobium genomes ranged from 82.95 to 83.76 %.The ANI coverage ranged from 6.2 to 52.7 %.The corresponding graphical output of ANI pairwise values from pyani is shown in Fig. S3.The dDDH    Planococcus halocryophilus DSM 24743 T .The position of APC 4016 T within this tree indicates that it is a separate species among these strains.These results show that although APC 4016 T shared a high 16S rRNA gene sequence % identity to the reference strains, their genomes were not highly similar.

Cell morphology, biochemical and phenotypic characterization
Cells of strain APC 4016 T are aerobic, motile cocci, 0.6-0.9µm in diameter, occur in clusters of two to eight (Fig. 3) and are Gram-stain positive to variable (Fig. S5).Motility was observed as tumbling and spinning motions.After culturing on MA for 3 days, colonies of strain APC 4016 T appeared smooth circular and light orange with darker coloured edges, and 1-1.2 mm in diameter.Colonies cultured on TSA were paler in colour and slightly crateriform (Fig. 4).Flagella were not observed in SEM imaging (Fig. 3).The colony morphology after 5 days of incubation is shown in Fig. 4. From the API ZYM assay, strain APC 4016 T was positive for alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase and α-glucosidase activity; weakly positive for valine arylamidase, cystine arylamidase, acid phosphatase and β-galactosidase, and negative for lipase, trypsin, α-galactosidase, β-glucuronidase, β-glucosidase, mannosidase and fucosidase activity.Strain APC 4016 T was susceptible to all of the tested antibiotics: ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), lincomycin (15 µg), neomycin (30 µg), novobiocin (5 µg), oleandomycin (15 µg), penicillin G (10 U), polymyxin B (300 U), rifampcin (30 µg), streptomycin (10 µg) and tetracycline (30 µg).The antibiotic susceptibility profile of strain 4016 T with corresponding zone of inhibition measurements is given in Table S2.Due to the lack of CLSI guidelines and breakpoint data for the genus Planococcus, susceptibility and resistance were interpreted as no growth or growth.We also note the lack of standardized antibiotic susceptibility testing methods for members of this genus in the literature, such as the use of alternative media instead of Mueller-Hinton, and  varying growth temperatures.This represents a deficiency in the standardization within this group; however, we have reported our results in line with existing literature.
Differential characteristics between APC 4016 T and the reference Planococcus/Planomicrobium strains are provided in Table 3.All the data for strain APC 4016 T is from this study, the data for the reference strains was acquired from the respective reference papers referred to in the Table 3 description.In the phenotypic assays, APC 4016 T shared a number of characteristics that are in accordance with members of the genus Planococcus, such as positive for catalase, negative for urease and hydrolysis of starch and a predominance of menaquinones 7 and 8 as the cellular respiratory quinones [2].However, strain APC 4016 T differed in that it was positive for nitrate Table 3. Differential characteristics of APC 4016 T and the reference Planococcus/Planomicrobium type strains Strains: 1, APC 4016 T (data from this study); 2, P. okeanokoites IFO 12536 T (data from [2,4] and [23]) ; 3, P. plakortidis DSM 2399 T (data from [51]); 4, P. salinarum DSM 23820 T (data from [3,52]); 5, P. donghaensis DSM 22276 T (data from [15]); 6, P. halotolerans SCU63 T (data from [53]).+, Positive; −, negative; w, weakly positive; nd, not determined; V, variable.reduction and hydrolysis of Tween 80, weakly positive for hydrolysis of aesculin and contained menaquinones MK5 and MK6, as well as MK7 and MK8.Strain APC 4016 T demonstrated a relatively wide temperature range for growth of 4-40 °C, similar to the reference strains (Table 3).Strain APC 4016 T also demonstrated relatively wide %NaCl and pH ranges for growth of 0-12 % (optimum 1 %) and pH 5.5-12 (inclusive; optimum, pH 7-7.5), respectively.Growth above pH 12 could not be determined due to instability of the growth media at higher pH.In contrast to the majority of the closely related reference strains, APC 4016 T was negative for oxidase and hydrolysis of casein but positive for β-galactosidase.
The overall cellular fatty acid composition profiles of strain APC 4016 T and the reference Planococcus/Planomicrobium strains were similar (Table 4).Strain APC 4016 T differed slightly in that it had very few straight-chain fatty acids and did not contain iso-C 16 : 0 as a major component (>10 %) but contained a significant proportion of a unique summed feature, iso-C 17 : 1 I/anteiso B (which could not be distinguished by the midi system).The cellular polar lipids of strain APC 4016 T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and three unknown lipids (Fig. S6).Based on the combination of phylogenetic distance, phenotypic and biochemical characteristics, strain APC 4016 T represents a novel species for which we propose the name Planococcus notacanthi sp.nov.
The type strain is APC 4016 T (=DSM 115753 T =NCIMB 15463 T ), isolated from the skin of a deep-sea snub-nosed spiny eel, Notacanthus chemnitzii, from a depth of 1000 m in the Northwest Atlantic Ocean (43.282N 49.121 W).The genome of the type strain is 3.6 Mb with a DNA G+C content of 45.25 mol%.
The GenBank accession numbers for the 16S rRNA gene sequence and the draft genome of strain APC 4016 T are OQ456996 and JASDCQ000000000, respectively.

Fig. 1 .
Fig. 1.Overview of the subsystem features of the draft genome of strain APC 4016 T based on the rast annotation and seed database.The counts of features per subsystem category are given.The total number of subsystem features was 1603.

(Fig. 2 .
Fig. 2. Maximum-likelihood evolutionary tree of distances based on 16S rRNA gene sequences, showing the relationship of strain APC 4016 T and related species.Bootstrap values based on 1000 replicates (>50 %) are shown.The tree was rooted by 16S rRNA gene sequences from Psychrobacillus lasiicapitis NEAU-3TGS17 and Sporosarcina saromensis HG645 of the family Caryophanaceae.

Fig. 4 .
Fig. 4. Colonies of APC 4016 T after culturing at 25 °C for 5 days on marine agar (a) and tryptic soy agar (b).

Table 1 .
General characteristics of the draft genome of strain APC 4016 T

Table 2 .
Summary of genomic comparisons between APC 4016 T and closely related type strains dDDH, digital DNA-DNA hybridization; ANI, average nucleotide identity; coverage, ANI alignment coverage.